Redox cycling of resorufin catalyzed by rat liver microsomal NADPH-cytochrome P450 reductase

The O-dealkylation of 7-alkoxyresorufins to the highly fluorescent compound, resorufin (7-hydroxyphenoxazone), provides a rapid, sensitive, and convenient assay of certain forms of liver microsomal cytochrome P450. The results of this study indicate that NADPH-cytochrome P450 reductase catalyzes the reduction of resorufin (and the 7-alkoxyresorufins) to a colorless, nonfluorescent compound(s). The reduction of resorufin by NADPH-cytochrome P450 reductase was supported by NADPH but not NADH, and was not inhibited by dicumarol, which established that the reaction was not catalyzed by contaminating DT-diaphorase (NAD[P]H-quinone oxidoreductase). In addition to the rate of reduction, the extent of reduction of resorufin was dependent on the concentration of NADPH-cytochrome P450 reductase. The maintenance of steady-state levels of reduced resorufin required the continuous oxidation of NADPH, during which molecular O2 was consumed. When NADPH was completely consumed, the spectroscopic and fluorescent properties of resorufin were fully restored. These results indicate that the reduction of resorufin by NADPH-cytochrome P450 reductase initiates a redox cycling reaction. Stoichiometric measurements revealed of 1:1:1 relationship between the amount of NADPH and O2 consumed and the amount of H2O2 formed (measured fluorometrically). The amount of O2 consumed during the redox cycling of resorufin decreased approximately 50% in the presence of catalase, whereas the rate of O2 consumption decreased in the presence of superoxide dismutase. These results suggest that, during the reoxidation of reduced resorufin, O2 is converted to H2O2 via superoxide anion. Experiments with acetylated cytochrome c further implicated superoxide anion as an intermediate in the reduction of O2 to H2O2. However, the ability of reduced resorufin to reduce acetylated cytochrome c directly (i.e., without first reducing O2 to superoxide anion) precluded quantitative measurements of superoxide anion formation. Superoxide dismutase, but not catalase, increased the steady-state level of reduced resorufin and considerably delayed its reoxidation. This indicates that superoxide anion is not only capable of reoxidizing reduced resorufin, but is considerably more effective than molecular O2 in this regard. Overall, these results suggest that NADPH-cytochrome P450 reductase catalyzes the one-electron reduction of resorufin (probably to the corresponding semiquinoneimine radical) which can either undergo a second, one-electron reduction (presumably to the corresponding dihydroquinoneimine) or a one-electron oxidation by reducing molecular O2 to superoxide anion.(ABSTRACT TRUNCATED AT 400 WORDS)     

A long-wavelength fluorescent substrate for continuous fluorometric determination of cellulase activity: resorufin-beta-D-cellobioside

A simple and reliable continuous assay procedure for measurement of cellulase activity from several species using the new substrate resorufin-beta-D-cellobioside (Res-CB) has been developed. The product of enzyme reaction, resorufin, exhibits fluorescence emission at 585 nm with excitation at 571 nm and has a pK(a) of 5.8, which allows continuous measurement of fluorescence turnover at or near physiological pH values. The assay performed using purified cellulase from the microscopic fungus Trichoderma reesei has been shown to give the kinetic parameters K(m) of 112 microM and V(max) of 0.000673 micromol/mL/min. Methods for performing the assay using cellulases isolated from both live Arabidopsis thaliana plant and Aspergillus niger fungal species are presented.     

Resorufin inhibits the in vitro metabolism and mutagenesis of benzo(a)pyrene

7-Hydroxyphenoxazin-3-one, commonly known as resorufin, strongly inhibits benzo(a)pyrene-induced mutation in the Ames bacterial reversion assay. The antimutagenic mechanism is due in part to redox cycling of resorufin with the concommitant transfer of reducing equivalents from NADPH to molecular oxygen. The diversion of electrons from cytochrome P-450 enzymes results in a large decrease in the percent of benzo(a)pyrene metabolized by rat liver microsomes as measured by HPLC. Resorufin stimulated a non-stoichiometric consumption of NADPH and was reduced in S-9 or microsomal solutions. These processes were sensitive to dicumarol and NADP inhibition to different degrees in each liver fraction. This suggests two pathways are involved in resorufin redox cycling, one involving DT-diaphorase and the other with NADPH cytochrome P-450 reductase. Oxygen was shown to be an electron acceptor for S-9 mediated resorufin redox cycling, but was not consumed by a microsomal solution in the presence of resorufin and NADPH.     

Thioredoxin catalyzes the reduction of insulin disulfides by dithiothreitol and dihydrolipoamide.

Thioredoxin from Escherichia coli was shown to catalyze the reduction of insulin disulfides by dithiothreitol. A quantitative assay was developed which measures the rate of insulin reduction spectrophotometrically at 650 nm as turbidity formation from the precipitation of the free insulin B chain. Thioredoxin, at 5 microM concentration, accelerated the reaction between 0.130 mM insulin and 1.0 mM dithiothreitol at pH 7 around 20-fold. The pH optimum of the reaction was 7.5. Thioredoxins from E. coli and calf liver showed similar specific activities. Stopped flow fluorescence measurements of the rate of reduction of thioredoxin-S2 by dithiothreitol showed a second order rate constant of 1647 M-1 s-1 at pH 7.2. This is between 10(2) to 10(3) times larger than the reaction between insulin or linear model disulfides and dithiothreitol. It is consistent with a ping-pong mechanism of thioredoxin catalysis since reduced thioredoxin is known to react very fast with insulin. Thioredoxin also catalyzed lipoamide-dependent reduction of the insulin disulfides in a coupled system with NADH, lipoamide, and lipoamide dehydrogenase. The fast spontaneous reaction between dihydrolipoamide and thioredoxin-S2 provides a mechanism for NADH or pyruvate-dependent disulfide reduction. The implication of the dithiol-disulfide oxidoreductase activity of thioredoxin for the regulation of enzyme activities by thiol oxidation-reduction control is discussed.     

Characterization of FMN-dependent NADH-quinone reductase induced by menadione in Escherichia coli

It was found that when Escherichia coli is grown in the presence of 0.2-0.3 mM menadione (2-methyl-1,4-naphthoquinone), an FMN-dependent NADH-quinone reductase increases more than 20-fold in the cytoplasmic fraction. The menadione-induced quinone reductase was isolated from the cytoplasmic fraction of induced cells. The purified enzyme had an Mr of 24 kDa on SDS-polyacrylamide gel electrophoresis. The enzyme required flavin as a cofactor and a half-maximum activity was obtained with 0.54 microM FMN or 16.5 microM FAD. The enzyme had a broad pH optimum at pH 7.0-8.0 and reacted with NADH, but not with NADPH. The reaction followed a ping-pong mechanism and the intrinsic Km values for NADH and menadione were estimated to be 132 microM and 2.0 microM, respectively. Dicoumarol was a simple competitive inhibitor with respect to NADH with a Ki value of 0.22 microM. The electron acceptor specificity of this enzyme was very similar to that of NAD(P)H: (quinone acceptor) oxidoreductase (EC 1.6.99.2, DT-diaphorase) from rat liver. Since menadione is reduced by the two-electron reduction pathway to menadiol, the induction of this enzyme is likely to be an adaptive response of E. coli to partially alleviate the toxicity of menadione.     

An essential histidine residue in the catalytic mechanism of mammalian glutathione reductase

Glutathione reductase from human erythrocytes was inactivated by ethoxyformic anhydride, and > 95% activity was lost by modification of about 1–1.5 histidine residues per flavin (or subunit), as measured by the increased absorbance at 240 nm. Full reactivation was obtained with hydroxylamine. The rate of inactivation increased with pH and an apparent pK = 5.9 was obtained for the protolytic dissociation. The modified enzyme was inactive with NADPH and GSSG as substrates, but almost fully active in catalysis of a transhydrogenase reaction involving pyridine nucleotides. The visible absorption spectrum of oxidized or two-electron-reduced enzyme was not changed, but the flavin fluorescence of oxidized enzyme increased 2-fold after the modification. NADPH or NADP⁺ did not protect the enzyme against inactivation. It is concluded that the modification affects a histidine involved in the second half-reaction of the catalysis, i.e. reduction of GSSG by the dithiol of reduced enzyme. Glutathione reductase from three additional mammalian sources was similarly inactivated, but enzyme from yeast was much less inactivated by the corresponding treatment with ethoxyformic anhydride.     

Expression of mammalian DT-diaphorase in Escherichia coli: purification and characterization of the expressed protein.

A full-length cDNA clone, pKK-DTD4, complementary to rat liver cytosolic DT-diaphorase [NAD(P)H:quinone oxidoreductase (EC 1.6.99.2)] mRNA was expressed in Escherichia coli. The pKK-DTD4 cDNA was obtained by extending the 5'-end sequence of a rat liver DT-diaphorase cDNA clone, pDTD55, to include an ATG initiation codon and the NH2-terminal codons using polymerase chain reaction (PCR). Restriction sites for EcoRI and HindIII were incorporated at the 5'- and 3'-ends of the cDNA, respectively, by the PCR reaction. The resulting full-length cDNA was inserted into an expression vector, pKK2.7, at the EcoRI and HindIII restriction sites. E. coli strain AB1899 was transformed with the constructed expression plasmid, and DT-diaphorase was expressed under the control of the tac promotor. The expressed DT-diaphorase exhibited high activity of menadione reduction and was inhibited by dicumarol at a concentration of 10(-5)M. After purification by Cibacron Blue affinity chromatography, the expressed enzyme migrated as a single band on 12.5% sodium dodecyl sulfate-polyacrylamide gel with a molecular weight equivalent to that of the purified rat liver cytosolic DT-diaphorase. The purified expressed protein was recognized by polyclonal antibodies against rat liver DT-diaphorase on immunoblot analysis. It utilized either NADPH or NADH as electron donor at equal efficiency and displayed high activities in reduction of menadione, 1,4-benzoquinone, and 2,6-dichlorophenolindophenol which are typical substrates for DT-diaphorase. The expressed DT-diaphorase exhibited a typical flavoprotein spectrum with absorption peaks at 380 and 452 nm. Flavin content determination showed that it contained 2 mol of FAD per mole of the enzyme. Edman protein sequencing of the first 20 amino acid residues at the NH2 terminus of the expressed protein indicated that the expressed DT-diaphorase is not blocked at the NH2 terminus and has an alanine as the first amino acid. The remaining 19 amino acid residues at the NH2 terminus were identical with those of the DT-diaphorase purified from rat liver cytosol.     

Rapid analysis of human fecal bile acids

A rapid, accurate, precise method for determining human fecal bile acids is reported. Feces are homogenized and then briefly extracted with boiling absolute ethanol. A portion of the extract is evaporated to dryness and the residue heated with mild alkali to hydrolyze bile acid 3α-hydroxyl esters. Aliquots of hydrolyzed crude extract are treated with resazurin reagent which effects a series of enzyme catalyzed reactions in which bile acid free 3α-hydroxyls are first oxidized to 3-oxo-groups in a reaction catalyzed by 3α-hydroxysteroid dehydrogenase. Resulting protons are transferred to β-nicotinamide adenine dinucleotide, yielding reduced β-nicotinamide adenine dinucleotide (β-NADH). β-NADH then reduces nonfluorescent resazurin to fluorescent resorufin in a reaction catalyzed by diaphorase. Developed fluorescence, which is proportional to the extract aliquots bile acid content, is excited at 565 nm and read at 580 nm, wavelengths which lie in a spectral region in which there is minimal fecal pigment absorption. 3-Oxo-bile acids and bile acid 3α-sulfates are extracted in the procedure but reduction and/or solvolysis is necessary before quantification.     

Delayed fluorescence from Rhodopseudomonas sphaeroides following single flashes.

Delayed fluorescence from Rhodopseudomonas sphaeroides chromatophores was studied with the use of short flashes for excitation. Although the delayed fluorescence probably arises from a back-reaction between the oxidized reaction center bacteriochlorophyll complex (P+) and the reduced electron acceptor (X-), the decay of delayed fluorescence after a flash is much faster (tau1/2 approximately 120 mus) than the decay of P+X-. The rapid decay of delayed fluorescence is not due to the uptake of a proton from the solution, nor to a change in membrane potential. It correlates with small optical absorbance changes at 450 and 770 nm which could reflect a change in the state of X-. The intensity of the delayed fluorescence is 11-18-fold greater if the excitation flashes are spaced 2 s apart than it is if they are 30 s apart. The enhancement of delayed fluorescence at high flash repetition rates occurs only at redox potentials which are low enough (less than +240 mV) so that electron donors are available to reduce P+X- to PX- in part of the reaction center population. The enhancement decays between flashes as PX- is reoxidized to PX, as measured by the recovery of photochemical activity. Evidently, the reduction of P+X- to PX- leads to the storage of free energy that can be used on a subsequent flash to promote delayed fluorescence. The reduction of P+X- also is associated with a carotenoid spectral shift which decays as PX- is reoxidized to PX. Although this suggests that the free energy which supports the delayed fluorescence might be stored as a membrane potential, the ionophore gramicidin D only partially inhibits the enhancement of delayed fluorescence. With widely separated flashes, gramicidin has no effect on delayed fluorescence. At redox potentials low enough to keep X fully reduced, delayed fluorescence of the type described above does not occur, but one can detect weak luminescence which probably is due to phosphorescence of a protoporphyrin.     

A long-wavelength fluorescent substrate for continuous fluorometric determination of α-mannosidase activity: Resorufin α-d-mannopyranoside

A simple and reliable continuous assay for measurement of α-mannosidase activity is described and demonstrated for analysis with two recombinant human enzymes using the new substrate resorufin α-d-mannopyranoside (Res-Man). The product of enzyme reaction, resorufin, exhibits fluorescence emission at 585 nm with excitation at 571 nm and has a pK_a of 5.8, allowing continuous measurement of fluorescence turnover at or near physiological pH values for human lysosomal and Drosophila Golgi α-mannosidases. The assay performed using recombinant Drosophila Golgi α-mannosidase (dGMII) has been shown to give the kinetic parameters K_m of 200 μM and V_max of 11 nmol/min per nmol dGMII. Methods for performing the assay using several concentrations of the known α-mannosidase inhibitor swainsonine are also presented, demonstrating a potential for use of the assay as a simple method for high-throughput screening of inhibitors potentially useful in cancer treatment.     

The soluble cytochromes c of methanol-grown Hyphomicrobium X. Evidence against the involvement of autoreduction in electron-acceptor functioning of cytochrome cL.

Hyphomicrobium X, grown on methanol with O2 or nitrate as electron acceptor, contains two major soluble cytochromes c. These were isolated in electrophoretically homogeneous form. They are related to cytochromes c already described for other methylotrophic bacteria and designated cytochromes cH and cL (properties indicated in that order) in view of the following characteristics: absorption maxima of the reduced forms (414, 520 and 551 nm and 414, 520 and 550 nm); molar absorption coefficients of the alpha-bands (23,700 M-1.cm-1 and 21,600 M-1.cm-1); maxima of the alpha-bands (no splitting) at 77 K (547.6 nm and 548.5 nm); Mr values of the native proteins (15,000 and 19,500); pI values (7.4 and 7.5, and 4.3); midpoint potentials at pH 7.0 (+292 mV and +270 mV). Both were monomers containing 1 haem c group per protein molecule, the oxidized forms binding cyanide at high pH. Autoreduction also occurred at high pH but at a rate significantly lower than that reported for other ferricytochromes c. On the other hand, the reverse situation applies to the reduction of ferricytochrome cL by reduced methanol dehydrogenase, the reduction occurring instantaneously at pH 7 but much more slowly at pH 9 (ferricytochrome cH was reduced at a 7-fold lower rate, but the rates at pH 7 and 9 were similar). Insignificant reduction was observed with cyclopropanol-inactivated enzyme or with enzyme in the presence of EDTA. In view of the dissimilarities, it is concluded that different mechanisms operate in the autoreduction of ferricytochrome cL and in its reduction by reduced methanol dehydrogenase.     

Evidence for two genetically distinct DNA primase activities specified by plasmids of the B and I incompatibility groups.

Soluble formate dehydrogenase from Methanobacterium formicicum was purified 71-fold with a yield of 35%. Purification was performed anaerobically in the presence of 10 mM sodium azide which stabilized the enzyme. The purified enzyme reduced, with formate, 50 mumol of methyl viologen per min per mg of protein and 8.2 mumol of coenzyme F420 per min per mg of protein. The apparent Km for 7,8-didemethyl-8-hydroxy-5-deazariboflavin, a hydrolytic derivative of coenzyme F420, was 10-fold greater (63 microM) than for coenzyme F420 (6 microM). The purified enzyme also reduced flavin mononucleotide (Km = 13 microM) and flavin adenine dinucleotide (Km = 25 microM) with formate, but did not reduce NAD+ or NADP+. The reduction of NADP+ with formate required formate dehydrogenase, coenzyme F420, and coenzyme F420:NADP+ oxidoreductase. The formate dehydrogenase had an optimal pH of 7.9 when assayed with the physiological electron acceptor coenzyme F420. The optimal reaction rate occurred at 55 degrees C. The molecular weight was 288,000 as determined by gel filtration. The purified formate dehydrogenase was strongly inhibited by cyanide (Ki = 6 microM), azide (Ki = 39 microM), alpha,alpha-dipyridyl, and 1,10-phenanthroline. Denaturation of the purified formate dehydrogenase with sodium dodecyl sulfate under aerobic conditions revealed a fluorescent compound. Maximal excitation occurred at 385 nm, with minor peaks at 277 and 302 nm. Maximal fluorescence emission occurred at 455 nm.     

DT-diaphorase-catalyzed two-electron reduction of quinone epoxides

DT-diaphorase catalyzes the two-electron reduction of the unsubstituted quinone epoxide, 2,3-epoxy-p-benzoquinone, at expense of NAD(P)H with formation of 2-OH-p-benzohydroquinone as the reaction product. The further conversion reactions of 2-OH-p-benzohydroquinone are influenced by the presence of O2 in the medium. Under aerobic conditions, 2-OH-p-benzohydroquinone undergoes autoxidation--probably with formation of 2-OH-semiquinone intermediates--to 2-OH-p-benzoquinone. The latter product is rapidly reduced by DT-diaphorase and, thus, its accumulation can be only observed upon exhaustion of NADPH. Under anaerobic conditions, 2-OH-p-benzohydroquinone does not undergo autoxidation and its accumulation is stoichiometrically (1:1) related to the amount of NADPH oxidized and epoxide substrate reduced. DT-diaphorase also catalyzes the reduction of the disubstituted quinone epoxide, 2,3-dimethyl-2,3-epoxy-1,4-naphthoquinone. Neither the aliphatic epoxide, trans-stilbene oxide, nor the aromatic epoxide, 4,5-epoxy-benzo[a]pyrene are substrates for DT-diaphorase. The reduction of 2,3-epoxy-p-benzoquinone is also catalyzed by the one-electron transfer enzyme, NADPH-cytochrome P450 reductase at a rate similar to that found with DT-diaphorase. However, this reaction differs from that catalyzed by DT-diaphorase in the distribution of molecular products as well as in the relative contribution of nonenzymatic reactions, i.e. semiquinone disproportionation and autoxidation.     

Plastocyanin conformation. The effect of nitrotyrosine modification and pH

Plastocyanin isolated from several species including spinach, poplar, and lettuce showed conformational changes both upon reduction and upon lowering the pH as determined by near-ultraviolet absorption and fluorescence measurements. The fluorescence excitation maximum was at 278 nm for all species of plastocyanin measured. In the case of spinach, the emission maximum was at 310–312 nm, similar to a tyrosine residue in solution. The fluorescence intensity increased 22% upon reduction of plastocyanin at pH 7.0. In poplar plastocyanin, the emission maximum was shifted to 335 nm and increased only 10% upon reduction. The 335 nm emission peak observed in poplar plastocyanin is attributed to Tyr 80 which is hydrogen bonded to a carbonyl group on the protein backbone. Tyr 83 was also shown to undergo fluorescence changes upon reduction since the redox state-dependent fluorescence changes decreased for a nitrotyrosine (nitrotyrosine-plastocyanin) derivative of this residue. These results show that the east face of the molecule, which contains both Tyr 80 and 83 as well as a possible binding site [1,2], undergoes conformational changes upon reduction. These conformational changes may be involved in promoting smooth electron transport between plastocyanin and its reaction partners. Both the absorption and fluorescence were found to be pH dependent. The quantum yield for fluorescence increased sharply below pH 6 for both oxidized and reduced spinach plastocyanin. This may be related to the appearance of a redox-inactive form of reduced plastocyanin [3]. The conformational changes observed at low pH may provide a mechanism for control of electron transport by the proton gradient. Low concentrations of CaCl₂ (10 mM) had no effect on plastocyanin fluorescence. However, addition of 2.7 M (NH₄)₂SO₄ eliminated the redox-dependent fluorescence changes.     

Fluorescence-microscopic measurement of intracellular cytochrome P-450 enzyme activity (ethoxyresorufin O-de-ethylation) in unfixed liver section.

A fluorescence-microscopic method has been developed for measurement of the intracellular kinetics of the cytochrome P-450 reaction, ethoxyresorufin O-de-ethylation, in individual hepatocytes in unfixed non-frozen liver sections obtained from control or 3-methylcholanthrene-pretreated mice. De-ethylation was enhanced in the presence of salicylamide, but inhibited in the presence of alpha-naphthoflavone, A total of 21 reaction rate curves (fluorescence of the metabolite, resorufin, versus time) were constructed for different individual hepatocytes and groups of two or three hepatocytes, in a total of 12 sections from four control and eight 3-methylcholanthrene-induced mice. No two reaction curves were identical, but the curves were classified, by similarity of curve shape and fluorescence intensity, into three control categories and four 3-methylcholanthrene-induced categories. It was considered that the data represented a total of seven types of hepatocytes, which differed in their ethoxyresorufin de-ethylase reaction characteristics. Hepatocytes in the sections from 3-methylcholanthrene-pretreated mice showed higher de-ethylase activities than the hepatocytes from control mice, but the increase in activity covered a wide range of values, from 3-fold to 17-fold higher than the mean control value, suggesting that 3-methylcholanthrene did not induce ethoxyresorufin de-ethylation to the same extent in all hepatocytes.     

Action mechanism of Escherichia coli DNA photolyase. II. Role of the chromophores in catalysis

DNA photolyase repairs pyrimidine dimers in DNA in a reaction that requires visible light. Photolyase from Escherichia coli is normally isolated as a blue protein and contains 2 chromophores: a blue FAD radical plus a second chromophore that exhibits an absorption maximum at 360 nm when free in solution. Oxidation of the FAD radical is accompanied by a reversible loss of activity which is proportional to the fraction of the enzyme flavin converted to FADox. Quantitative reduction of the radical to fully reduced FAD causes a 3-fold increase in activity. The results show that a reduced flavin is required for activity and suggest that flavin may act as an electron donor in catalysis. Comparison of the absorption spectrum calculated for the protein-bound second chromophore (lambda max = 390 nm) with fluorescence data and with the relative action spectrum for dimer repair indicates that the second chromophore is the fluorophore in photolyase and that it does act as a sensitizer in catalysis. On the other hand, enzyme preparations containing diminished amounts of the second chromophore do not exhibit correspondingly lower activity. This suggests that reduced flavin may also act as a sensitizer in catalysis. The blue color of the enzyme is lost upon reduction of the FAD radical. The fully reduced E. coli enzyme exhibits absorption and fluorescence properties very similar to yeast photolyase. This indicates that the two enzymes probably contain similar chromophores but are isolated in different forms with respect to the redox state of the flavin.     

Study of the redox properties of naphthazarin (5,8-dihydroxy-1,4-naphthoquinone) and its glutathionyl conjugate in biological reactions: one- and two-electron enzymatic reduction

Naphthazarin (5,8-dihydroxy-1,4-naphthoquinone), the basic unit of several tetracyclic antitumor antibiotics, and its glutathione conjugate were reduced by the one- and two-electron transfer flavoproteins NADPH-cytochrome P450 reductase and DT-diaphorase to their semi- and hydroquinone forms, respectively. Kinetic studies performed on purified DT-diaphorase showed the following results: KNADPHm = 68 microM, KQuinonem = 0.92 microM, and Vmax 1300 nmol X min-1 X microgram enzyme-1. Similar studies performed on purified NADPH-cytochrome P450 reductase indicated a lower KNADPHm (10.5 microM) and higher KQuinonem (2.3 microM). The Vmax values were 20-fold lower (46 nmol X min-1 X micrograms enzyme-1) than those observed with DT-diaphorase. DT-diaphorase reduced the naphthazarin-glutathione conjugate with an efficiency 5-fold lower than that observed with the parent quinone. The nucleophilic addition of GSH to naphthazarin proceeded with GSH consumption at rates slower than those observed with 1,4-naphthoquinone and its monohydroxy derivative, 5-hydroxy-1,4-naphthoquinone. The initial rate of GSH consumption during these reactions did not vary whether the assay was carried out under anaerobic or aerobic conditions. Autoxidation accompanied the DT-diaphorase and NADPH-cytochrome P450 reductase catalysis of naphthazarin and its glutathionyl adduct as well as the 1,4-reductive addition of GSH to naphthazarin. Superoxide dismutase at catalytic concentrations (nM range) enhanced slightly (1.1- to 1.6-fold) the autoxidation following the enzymatic catalysis of naphthazarin. Autoxidation during the GSH reductive addition to 1,4-naphthoquinones decreased with increasing number of -OH substituents, 1,4-naphthoquinone greater than 5-hydroxy-1,4-naphthoquinone greater than 5,8-dihydroxy-1,4-naphthoquinone, thus revealing that the contribution of redox transitions other than autoxidation, e.g., cross-oxidation, to the decay of the primary product of nucleophilic addition increases with increasing number of -OH substituents. Superoxide dismutase enhanced substantially the autoxidation of glutathionyl-naphthohydroquinone adducts, thereby affecting only slightly the total GSH consumed and GSSG formed during the reaction. The present results are discussed in terms of the relative contribution of one- and two-electron transfer flavoproteins to the bioreductive activation of naphthazarin and its glutathionyl conjugate as well as the importance of autoxidation reactions in the mechanism(s) of quinone cytotoxicity.     

Modification of myosin subfragment 1 tryptophans by dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide

Modification of tryptophanyl residues (Trps) of myosin subfragments 1 (S-1) was performed with dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide (DHNBS). Under controlled conditions, pH 6 at 0 degrees C and 10-min reaction with 10-100-fold molar excess, K+(EDTA) activity was reduced down to less than half, whereas Ca2+-ATPase activity increased and acto-S-1-ATPase was not affected. The number of modified Trps (up to 2.5) agreed well with the number of 2-hydroxy-5-nitrobenzyl moieties incorporated in S-1. The thiol groups of S-1 were not affected up to 50-fold molar excess of DHNBS, thus indicating that the modification was selective for Trps. The modification of as few as one Trp caused a blue shift of the emission spectrum, accompanied by a reduction in the fluorescence quantum yield. The accessibility of Trps to the fluorescence quencher acrylamide is drastically reduced upon modification, indicating that DHNBS-reactive Trps are more "exposed" than the DHNBS-refractive ones. DHNBS modification did not seem to affect the ATP-induced tryptophan fluorescence enhancement of S-1. The effect of DHNBS modification of the intrinsic fluorescence of S-1 indicates that the modified Trps are located in a polar environment and that they may be identical with the long-lifetime Trps of Torgerson [Torgerson, P. (1984) Biochemistry 23, 3002-3007]. The most reactive Trp is located in the N-terminal 27-kDa fragment of the S-1 heavy chain. It might also be inferred from the above data that the nonexposed and ATP-perturbed Trp(s) is (are) located in the 50-kDa fragment.     

Measurement of EROD Activity: Caution on Spectral Properties of Standards Used

The activity of the enzyme 7-ethoxy-resorufin-O-deethylase (EROD) has been extensively used in biomonitoring studies for more than a decade. Although the analytical procedure is simple, it is often poorly characterized. In this study spectral properties of particular standard compounds used to measure EROD activity (ethoxyresorufin and resorufin, standards from Molecular Probes) were tested in order to optimize excitation and emission wavelengths to be used in the fluorimetric assay of EROD activity. The optimal excitation wavelength for the detection of resorufin was 560 nm. At this wavelength the excitation represents only 37% of its maximum level for ethoxyresorufin, while it represents 86% for resorufin. This allows discrimination between the fluorescence emitted by both standards, favoring the formed resorufin. Our results demonstrate that any analytical work using spectrofluorometry to measure EROD activity should be preceded by precise determination of the spectral characteristics of each set of standards used.     

Characterization of the Reduced Nicotinamide Adenine Dinucleotide Phosphate-Nitrate Reductase of Aspergillus nidulans

The reduced nicotinamide adenine dinucleotide phosphate (NADPH)-nitrate oxidoreductase (EC 1.6.6.2) from Aspergillus nidulans was purified over 200-fold by use of salt fractionation, gel filtration, and ion-exchange chromatography. The purified enzyme was specific for NADPH and catalyzed reduction of nitrate, cytochrome c from isolated mitochondria of Aspergillus, and mammalian cytochrome c. An S(0.725) (20, w) of 7.8 was derived with sucrose density gradient centrifugation, and a Stokes radius of 6.4 nm was derived by gel filtration on Sephadex G-200. From these values, a molecular weight of 197,000 was computed, assuming v = 0.725 cm(3)/g. The spectral properties of the purified enzyme suggested a flavine component was present but revealed no pattern indicative of a hemoprotein. A cytochrome c, similar to the cytochrome c from isolated mitochondria, was found unassociated with the nitrate reductase after ion-exchange chromatography. No NADPH-nitrate reductase activity was detected in isolated mitochondria. Spectrally discernable reduction of the flavine component of the enzyme at 450 nm was noted after reaction with NADPH. This reduction was inhibited by p-chloromercuribenzoate but not by KCN. The addition of nitrate to NADPH reduced enzyme caused a reoxidation of the flavine component via a reaction which was inhibited by KCN but not by p-chloromercuribenzoate. The half-life of the purified enzyme at 37 C was 20 min for NADPH-nitrate reductase and 35 min for NADPH-cytochrome c reductase.